Urinary TGF-alpha, EGF, and TGF-beta were efficiently concentrated on microparticulate silica and separated by acetonitrile elution. Further resolution was obtained by sequential chromatography based on molecular size (Bio-Gel), charge (CM-cellulose), and hydrophobicity (RP-HPLC). The high molecular weight (HMW) TGF of 30,000 to 35,000 Mr, previously reported in the urine of various cancer patients is, in patients with malignant astrocytomas, indistinguishable from the HMW form of hEGF in terms of apparent molecular size, EGF receptor binding activity, EGF immunoreactivity and clonogenic, activity. However, in comparison to bulk (25 liters) urine from normal individuals, equivalently large urine samples from these brain tumor patients contained about fourfold more HMW hTGF/hEGF. hTGF-alpha was not identified in either source of bulk urine. Secondly, in an in vitro study of HMW TGFs, conditioned medium of A673 cells (a human rhabdomyosarcoma cell line) was found to contain principal peaks of EGF radioreceptor and clonogenic activity in sodium dodecyl sulfate-polyacrylamide gel electrophoresis slices corresponding to Mr 15,000 and 22,000 in an RP-HPLC sample eluting at 25-26% acetonitrile, and two additional higher Mr activities in a 22-23% acetonitrile eluting region. Neither of these active regions from HPLC competed in radioimmunoassay under reduced and denatured conditions for hEGF or rTGF-alpha. Evaluation of TGF-alpha mRNA content in A673 cells is currently in progress. Thirdly, the pooled urine of patients with disseminated breast cancer contains immunoreactive TGF-alpha which is not present in comparable control urine from normal individuals. Fourthly, urinary proteins from individual 24-hour urine samples were concentrated, fractionated, and scored for immunoreactive TGF-alpha by RIA. The scattergram results, in order of decreasing nanograms of urinary TGF-alpha per gram creatinine, were samples from (1) patients with disseminated breast carcinoma and lactating females, (2) pregnant women, (3) patients with small primary breast cancers with no or minimal evidence of regional metastasis, and (4) healthy normal women.